乳腺癌干细胞的富集及相关基因表达

Enrichment of breast cancer stem cells and correlative gene expression

目的通过微球体培养富集乳腺癌干细胞,并检测其相关基因的表达。方法将MCF-7乳腺癌细胞进行第一代微球体培养,7d后消化微球体获得单细胞悬液,取部分细胞进行第二代微球体培养,另一部分细胞则在胶原底物进行分化培养。于培养第11天收集第二代微球体细胞、分化细胞,采用流式细胞术进行干细胞比例分析;同时应用Real-time PCR技术检测β-catenin、CXCR4、SOX2、ALDH3A1 mRNA表达。结果流式细胞分析显示,微球体细胞和分化细胞的侧亚群细胞比例分别为(7.36±0.54)%和(4.32±0.42)%(P<0.05),CD55high比例分别为(17.41±1.09)%和(4.47±0.33)%(P<0.05)。Real-time PCR检测结果表明,微球体细胞与分化细胞相比,β-catenin、CXCR4、SOX2及ALDH3A1 mRNA表达分别上调了约1.5、5.0、4.0和5.7倍(P<0.01)。结论微球体细胞富集了较高比例的乳腺癌干细胞,且β-catenin、CXCR4、SOX2、ALDH3A1 mRNA呈现高表达。

Objective To enrich breast cancer stem cells through culturing mammospheres,and to detect the correlative gene expression. Methods MCF-7 breast cancer cells were cultured in suspension to generate primary mammospheres.After 7 d,mammospheres were dissociated to yield single cells.An aliquot of the cells were cultured in suspension to generate secondary mammospheres,and the other ones were cultured on a collagen substratum to induce differentiation.After 11 d of cultivation,the proportion of cancer stem cells was measured in cells derived from secondary mammosphere cells or differentiated cells by flow cytometry,and the expressions of β-catenin,CXCR4,SOX2 and ALDH3A1 mRNA were detected by Real-time PCR. Results Flow cytometry analysis indicated that（7.36±0.54）% and（17.41±1.09）% of the mammosphere cells were side population and CD55high,respectively,compared with（4.32±0.42）% and（4.47±0.33）% for the differentiated cells（P〈0.05）.Real-time PCR analysis suggested that β-catenin,CXCR4,SOX2 and ALDH3A1 mRNA were expressed in the mammosphere cells at higher levels than the differentiated cells by about 1.5,5.0,4.0 and 5.7 fold,respectively（P〈0.01）. Conclusion Mammosphere cells contained higher propotion of breast cancer stem cells and expressed higher levels of β-catenin,CXCR4,SOX2 and ALDH3A1 mRNA.